Sigma-Aldrich Y. ENTEROCOLITICA MILLICHROME# PLUS AGAR

Step 1 (Preparation of 1L Y. enterocolitica Millichrome^™ plus Agar Base)

• Disperse slowly 41.3 g of powder base in 1 L of purified water.
• Stir until agar is well thickened.
• Heat and bring to boil (100 °C) while swirling or stirring regularly. DO NOT HEAT TO MORE THAN 100 °C. DO NOT AUTOCLAVE AT 121 °C.

Warning 1: If using an autoclave, do so without pressure. Advice 1: For the 100 °C heating step, mixture may also be brought to a boil in a microwave oven: after initial boiling, remove from oven, stir gently, then return to oven for short repeated bursts of heating until complete fusion of the agar grains has taken place (large bubbles replacing foam).

• Cool in a water bath to 45-50 °C +/- 2 °C.
• Step 2 (Preparation of the Y. enterocolitica Millichrome^™ plus Supplement)
• Prepare a stock solution of the supplement: Add 100 mg to 1 mL of purified water.
• Swirl well until complete dissolution. Filter sterilize at 0.45 μm.

Warning 2: This supplement stock solution should be used immediately after Preparation, or can be stored at -20 °C and used within 15 days.
Step 3 (Mixing of the prepared base and the prepared supplement)

• Add 1 mL of the prepared supplement solution to the prepared base cooled at 45-50 °C +/- 2 °C.
• Swirl gently to homogenize.
• Pour into sterile Petri dishes.
• Let it solidify and dry.

MilliChrome is a trademark of Merck KGaA, Darmstadt, Germany